Lipidated imidazoquinoline derivatives

ABSTRACT

The compounds of the subject invention are adjuvant molecules that comprise an imidazoquinoline molecule covalently linked to a phospho- or phosphonolipid group. The compounds of the invention have been shown to be inducers of interferon-a, IL-12 and other immunostimulatory cytokines and possess an improved activity profile in comparison to known cytokine inducers when used as adjuvants for vaccine antigens.

This application is a continuation application of U.S. patentapplication Ser. No. 13/125,342, to be issued Jan. 7, 2014 as U.S. Pat.No. 8,624,029, which was filed Apr. 21, 2011, pursuant to 35 USC 371 asa United States National Phase Application of International PatentApplication Serial No. PCT/US2009/061867 filed Oct. 23, 2009, whichclaims the benefit of U.S. Provisional 61/108,210 filed Oct. 24, 2008,U.S. Provisional 61/224,226 filed Jul. 9, 2009, and U.S. Provisional61/229,933 filed Jul. 30, 2009, the entire contents of each of which arehereby incorporated by reference.

BACKGROUND

The present invention relates to novel adjuvant compounds, processes fortheir preparation, compositions containing them, and their use asvaccine adjuvants.

The refinement and simplification of microbial vaccines and the use ofsynthetic and recombinant subunit antigens to improve vaccinemanufacturability and safety has resulted in a decrease in vaccinepotency. This has led to studies on the co-administration of adjuvantswith antigens to potentiate vaccine activity and the weak immunogenicityof synthetic and recombinant epitopes. Adjuvants are additives thatenhance humoral and/or cell mediated immune responses to a vaccineantigen. The design of vaccine adjuvants, however, has historically beendifficult because of the complex nature of the molecular mechanismsinvolved in immune system function. Although the addition of microbialcomponents has long been known to enhance adaptive immune responses,only recently was it shown that toll-like receptors (TLRs) on cellsinvolved in immune surveillance, such as epithelial and dendritic cells,engage many of these microbial products via so-called“pathogen-associated patterns” or PAMPs. Many vaccine adjuvants andstand-alone immunomodulators appear to interact with members of the TLRfamily.

Of the 10 known TLRs that have been identified in humans, five areassociated with the recognition of bacterial components (TLRs 1, 2, 4,5, 6) and four others (TLRs 3, 7, 8, 9) appear to be restricted tocytoplasmic compartments and are involved in the detection of viral RNA(TLRs 3, 7, 8) and unmethylated DNA (TLR9) (Iwasaki, A., Nat Immunol2004, 5, 987) Activation of TLRs regulates intracellular signalingpathways and leads to gene expression via interaction with intracellularadapter molecules such as MyD88, TRIF, TIRAP, and TRAM (Akira, S. NatRev Immunol 2004, 4, 499; Takeda, K. Semin Immunol 2004, 16, 3). Theseadapter molecules can differentially regulate the expression ofinflammatory cytokines/chemokines and type I interferons (IFNa/b), whichcan lead to the preferential enhancement of antigen-specific humoral andcell-mediated immune responses (Zughaier, S. Infect Immun 2005, 73,2940). Humoral immunity is the major line of defense against bacterialpathogens, whereas the induction of cytotoxic T lymphocytes (CTLs)appears to be crucial for protective immunity in the case of viraldisease and cancer.

Currently, a group of aluminum salts known as alum are the dominantadjuvants used in human vaccines. But alum typically only enhanceshumoral (Th2) immunity and is generally used intramuscularly due tolocal toxicity by other routes (e.g., subcutaneous or intradermalinoculation leads to granulomas) (Aguilar, J. Vaccine 2007, 25, 3752).Other potential side effects of alum include increased IgE production,allergenicity and neurotoxicity. Thus, new safe and effective vaccineadjuvants are needed which are able to stimulate both antibody andTh1-type immune responses and that are compatible with different routesof administration and antigen formulations.

In the case of TLR7 and TLR8 activation, a few different classes ofsmall molecule mimetics of the natural (U- and/or G-rich) viral ssRNAligands have been identified. These include certain antiviral compoundsrelated to oxidized guanosine metabolites (oxoguanosines), whichprimarily interact with TLR7 (Heil, F. Eur J Immunol 2003, 33, 2987;Hemmi, 2002) and derivatives of adenine which engage TLR7 and/or TLR8.The immune stimulating ability of these compounds has been attributed tothe TLR/MyD88-dependent signaling pathways and the production ofcytokines, including IL-6 and type I (particularly interferon-a) and IIinterferons. TLR7 or TLR8 activation leads to the upregulation ofco-stimulatory molecules (e.g. CD-40, CD-80, CD-86) and class I and IIMHC molecules on dendritic cells (DCs). DCs are the principal cells ofthe immune system involved in uptake and presentation of antigens to Tlymphocytes. Plasmacytoid dendritic cells (pDCs), which preferentiallyexpress TLR7, are professional interferon-a producing cells; whereasmDCs express TLR8 only. TLR8 activation on mDCs leads to thepreferential production of pro-inflammatory cytokines such as IL-12,TNF-a, and IFN-g and cell-mediated immunity (CMI).

One class of adenine derivatives that has received a considerable amountof attention are the 1H-imidazo[4,5-c]quinolines (IQs). The prototypicalmember of this class imiquimod (R847, S-26398) was found to be effectiveagainst genital papilloma virus infections, actinic keratosis, and basalcell carcinoma when applied topically in cream form. However, imiquimodhas relatively low interferon-inducing activity and both oral andtopical preparations are not without side-effects. In fact, serious sideeffects were reported in an HCV clinical trial with imiquimod. The largeimmunological “footprint” of TLR7 agonists in general has led concernsover toxicity: Clinical trials with another TLR7 agonist ANA-975, anoxoguanosine derivative, were recently suspended due to toxicity issues.

Another member of the IQ class of TLR7/8 ligands and a derivative of ametabolite of imiquimod is resiquimod. Resiquimod (R-848, S-28609) alsoactivates TLR7 in macrophages and DCs in a MyD88-dependent manner eitherdirectly or indirectly via an accessory molecule and upregulatesco-stimulatory molecules and MHCI/II in DCs. But in contrast toimiquimod, the more potent and toxic resiquimod is also a ligand forTLR8 signaling, which leads to the reversal of CD4+ regulatory (Treg)cell function. Using transfected HEK293 cells, it was recently shownthat TLR7 agonists are more effective at generating IFN-α andIFN-regulated cytokines, whereas TLR8 agonists were more effective atinducing proinflammatory cytokines such as TNF-α and IL-12, suggestingthat TLR7 activation may be more important for antibody responses(Th2-type responses) while TLR8 activation should drive CMI or Th1-typeimmune responses. However, as mentioned above, many TLR7/8 agonistsoften display toxic properties, are unstable, and/or have unsubstantialimmunostimulatory effects. Thus, the discovery and development ofeffective and safe adjuvants that activate TLR7 and/or TLR8 is essentialfor improving the efficacy and safety of existing and new vaccines viahelping to control the magnitude, direction, and duration of the immuneresponse against antigens.

Unlike TLR2 and TLR4, which recognize PAMPs on cell surfaces, TLR7/8PAMPs are sensed in the endosomal/lysosomal compartments and requireendosomal maturation. Cellular uptake is prerequisite for cellularactivation in the case of natural and zenobiotic TLR7/8 ligands such asimiquimod and resiquimod. Thus, strategies that would increase thepenetration of the TLR7/8 ligand into DCs and other immune cells couldenhance TLR activation and vaccine efficacy as well as ameliorate toxiceffects.

Lipid conjugates of nucleoside drugs are known in the art to enhanceoral bioavailability in general as well as permit incorporation of theresulting “nucleolipid” into lipid membranes of liposomes. Incorporatingunstable and/or toxic drugs in liposomes establishes a slow-releasecarrier system or molecular depot, which protects the drug fromdegradation and decreases toxic side effects. The potency of such “lipidprodrugs” has been reported to be comparable to that of thenon-derivatized drugs (U.S. Pat. No. 5,827,831—NeXstar). Depotpreparations of imidazoquinolines and fatty acylated IQs have beenreported in the art for the purposes of maintaining the IQ for anextended period within a localized tissue region to decrease metabolismand toxicity (WO 2005/001022—3M). However, conjugating animidazoquinoline to a phospho- or phosphonolipid in a specific manner inorder to facilitate uptake into immune cells, when administered alone orin depot formulation with an antigen, and enhance endosomal TLR7/8activation and antigen presentation is not known in the art. Enhancedimmune responses with compounds of the subject invention are possiblydue to direct interaction of compounds of formula (I) with endosomalTLR7 and/or TLR8 and/or interaction of an active metabolite afterenzymatic action.

BRIEF DESCRIPTION OF THE INVENTION

The compounds of the invention have been shown to be inducers ofinterferon-a, IL-12 and other immunostimulatory cytokines and maypossess an improved activity-toxicity profile in comparison to knowncytokine inducers when used as adjuvants for vaccine antigens in thetherapeutic or prophylactic treatment of infectious diseases and cancer.These compounds are also novel per se.

SUMMARY OF THE INVENTION

The compounds of the subject invention are adjuvant molecules thatcomprise a imidazoquinoline molecule which may be covalently linked to aphospho- or phosphonolipid group. The compounds of the subject inventionare broadly described by Formula I:

wherein

R₁=H, C₁₋₆ alkyl, C₁₋₆ alkylamino, C₁₋₆ alkoxy, C₃₋₆ cycloalkylC₁₋₆alkyl, C₃₋₆ cyclo alkylC₁₋₆alkylamino, C₃₋₆ cycloalkylC₁₋₆ alkoxy, C₁₋₆alkoxyC₁₋₆ alkyl, C₁₋₆ alkoxyC₁₋₆ alkylamino, C₁₋₆alkoxyC₁₋₆alkoxy;branched or unbranched and optionally terminally substituted with ahydroxyl, amino, thio, hydrazino, hydrazido, azido, acetylenyl,carboxyl, or maleimido group,

Z=C₂-C₆ alkyl or alkenyl, unsubstituted or terminally substituted by—(O—C₂-C₆alkyl)₁₋₆-

Y=O, NH

X=O, CH₂, CF₂

W=O or S

m=1-2,

wherein

R₂=H or straight/branched/unsaturated C₄-C₂₄ alkyl or acyl

R₃=straight/branched/unsaturated C₄-C₂₄ alkyl or acyl

R₄, R₅=independently H, C₁-C₆alkyl, C₁-C₆ alkoxy, halogen, ortrifluoromethyl; or taken together alternatively form a 6-membered aryl,heteroaryl containing one nitrogen atom, cycloalkyl, or heterocycloalkylring containing one nitrogen atom; unsubstituted or substituted by oneor more of C₁-C₆alkyl, C₁-C₆ alkoxy, halogen, or trifluoromethyl,

or pharmaceutically acceptable salts thereof.

In one embodiment, the compounds of the subject invention are morespecifically described by Formula II:

wherein

R₁=H, C₁₋₆alkyl, C₁₋₆alkylamino, C₁₋₆alkoxy, C₃₋₆cycloalkylC₁₋₆alkyl,C₃₋₆cycloalkylC₁₋₆alkylamino, C₃₋₆cycloalkylC₁₋₆alkoxy,C₁₋₆alkoxyC₁₋₆alkyl, C₁₋₆alkoxyC₁₋₆alkylamino, C₁₋₆alkoxyC₁₋₆alkoxy;branched or unbranched and optionally terminally substituted with ahydroxyl, amino, thio, hydrazino, hydrazido, azido, acetylenyl,carboxyl, or maleimido group,

n=1-6

Y=O, NH

X=O, CH₂, CF₂

W=O or S

m=1-2,

R₂=H or straight/branched/unsaturated C₄-C₂₄ alkyl or acyl

R₃=straight/branched/unsaturated C₄-C₂₄ alkyl or acyl (e.g.phosphatidyl, lysophosphatidyl ether or ester when W=O, X=O, m=1)

TABLE 1 Example Ref. No. R₁ n m 1 — — — — 2 — — — — 3 L1 H 2 1 4 L2 n-Bu2 1 5 L3 CH₂OEt 2 1 6 L4 CH₂OEt 4 1 7 — — — — 8 L5 CH₂OEt 2 2 9 — — — —For all Examples shown: Y = W = X = O; R₂ = R₃ = hexadecanoyl

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: schematic representation of study design

FIG. 2: graph showing p27-specific CD8 response

FIG. 3: graph showing p27-specific cytotoxicity detected in vivo

FIG. 4: graph showing antigen specific CD 4 T cell response

FIGS. 5A-5G Graphs showing serum cytokine response (IFNa, IL-12, IFNg,IL-6, TNFa, MCP-1 and MIG respectively) in groups immunized with theliposome-based formulations without (−) or with various amounts ofTLR7/8 ligands together with QS21 and MPL.

FIGS. 6A-6G Graphs showing serum cytokine response (IFNa, IL-12, IFNg,IL-6, TNFa, MCP-1 and MIG respectively) in groups immunized with theemulsion-based formulations without (−) or with various amounts ofTLR7/8 ligands together with QS21 and MPL.

DETAILED DESCRIPTION OF THE INVENTION Example 1 General procedure forthe preparation of4-Amino-1-[2-(1,2-dipalmitoyl-sn-glycero-3-phospho)alkyl]-1H-imidazo[4,5-c]quinolines(Compound (I), Y=W=X=O, m=1)

Imidazoquinoline monophosphate diglycerides V were prepared by coupling4-amino-1-hydroxyalkyl-imidazoquinolines III (Gerster et al. J Med Chem2005, 48, 3481; Izumi et al. Bioorg Med Chem 2003, 11, 2541) with 1-Hphosphonate IV according to methods known in the art (Crossman, et al. JChem Soc, Perkin Trans 1, 1997, 2769; Westerduin, et. al. Tet Lett,1986, 15, 6271; Nikolaev, et al., Carbohydr Res, 1990, 204, 65) asfollows: Imidazoquinoline III (1 eq) and H-phosphonate IV (2 eq) weresuspended in n-heptane and, after evaporation of the solvent, driedovernight under high vacuum. The resulting residue was dissolved inpyridine (0.01 M compound III), treated with pivaloyl chloride (12.4eq), and then stirred at room temperature for 6 h. A solution of iodine(4 eq) in 19:1 pyridine-water (0.04 M) was added and the resultingmixture stirred at room temperature for 1 h and then partitioned betweenCHCl₃ and 1 M aq Na₂S₂O₅. The layers were separated and the aqueouslayer was extracted twice with CHCl₃. The combined organic extracts werewashed with 1 M triethylammonium borate buffer (pH 8), dried (Na₂SO₄),and concentrated. The residue obtained was purified by flashchromatography on silica gel (gradient elution, 0→25% MeOH—CHCl₃) andthen by reverse phase chromatography (Bakerbond C8 in CH₃CN containing1% TEA, eluting with 0→60% MeOH—CH₃CN containing 1% Et₃N) to providecompound V as a colorless solid.

Example 2 Preparation of4-Amino-1-(4-hydroxybutyl)-2-ethoxymethyl-1H-imidazo[4,5-c]quinolinehydrochloride (Compound (III), R₁=CH₂OCH₂CH₃, n=4)

(1) A suspension of 4-hydroxy-3-nitroquinoline (Gerster et al. J MedChem 2005, 48, 3481) in DMF (0.7 M) was treated dropwise with POCl₃ (1.2eq) and stirred at 50° C. for 30 min. The reaction mixture was pouredinto ice-water and extracted twice with CH₂Cl₂. The combined organiclayers were washed with water, dried (Na₂SO₄) and concentrated. Thecrude product obtained was added to a solution of 4-amino-butanol (1.3eq) and triethylamine (1.9 eq) in EtOH and heated to reflux for 15 min.After concentration, flash chromatography on silica gel (gradientelution, 2→4% MeOH—CHCl₃) afforded4-(4-hydroxybutyl)amino-3-nitroquinoline as a yellow solid in 97% yield.

(2) A solution of the compound prepared in (1) above in EtOAc (0.1 M)was hydrogenated in the presence of 5% Pt/C (5% w/w) and MgSO₄ (1.5 eq)at 50 psig for 6 h. The reaction mixture was filtered through celite andconcentrated. The orange oil obtained was heated with ethoxyacetic acid(11 eq) at 150° C. for 1 h. The reaction mixture was cooled to 0° C.,basified to pH 10 with conc NH₄OH, and extracted twice with CH₂Cl₂. Thecombined organic layers were dried (Na₂SO₄) and concentrated. Flashchromatography on silica gel (1:60 MeOH—CHCl₃) gave the ethoxyacetatederivative which was treated with 2.6 M NaOH (5.0 eq) in EtOH (0.20 M)at room temperature for 1 h. Ethanol was removed under reduced pressureand the aqueous layer was extracted several times with AcOEt and CH₂Cl₂.The combined organic layers were dried (Na₂SO₄), and concentrated. Flashchromatography on silica gel (gradient elution, 1:50→1:15 MeOH—CHCl₃)afforded 1-(4-hydroxybutyl)-1H-imidazo[4,5-c]quinoline as a solid in 74%yield. ¹H NMR (CDCl₃, 400 MHz) δ 9.29 (s, 1H), 8.25 (dd, 2H), 7.67 (m,2H), 4.89 (s, 2H), 4.71 (t, 2H), 3.79 (m, 2H), 3.62 (dd, 2H), 2.12 (m,2H), 1.82 (m, 2H), 1.25 (t, 3H).

(3) A solution of the compound prepared in (2) above and peracetic acid(1.2 eq) in ethanol (0.4 M) was heated at 60° C. for 2.5 h. Afterconcentration, the crude product obtained was purified by chromatographyon silica gel (gradient elution, 1:30→1:6 MeOH—CHCl₃) to afford1-(4-hydroxybutyl)-1H-imidazo[4,5-c]quinoline 5-N-oxide as a yellowsolid in 94% yield

(4) A suspension of the compound prepared in (3) above in CH₂Cl₂ (0.43M) was treated with NH₄OH (30% aq solution, 2.7 mL) followed byp-toluenesulfonyl chloride (1.0 eq) dropwise. The resulting mixture wasstirred at room temperature for 1.5 h and then concentrated. Flashchromatography on silica gel (gradient elution, 1:30→1:9 MeOH—CHCl₃)afforded 4-amino-1-(4-hydroxybutyl)-1H-imidazo[4,5-c]quinoline as anorange solid in quantitative yield.

(5) A solution of the compound prepared in (4) above in dioxane (0.12M)at 50° C. was treated dropwise with 4N HCl in dioxane (1.5 eq) and thenallowed to cool to room temperature. The solid precipitate wascollected, washed with dioxane, and dried to give4-amino-1-(4-hydroxybutyl)-1H-imidazo[4,5-c]quinoline hydrochloride saltin 89% yield: ¹H NMR (CDCl₃-CD₃OD, 400 MHz) δ 8.13 (d, 1H), 7.97 (d,1H), 7.65 (t, 1H), 7.55 (t, 1H), 4.89 (bs, 2H), 4.68 (m, 2H), 3.75 (m,2H), 3.68 (dd, 2H), 2.10 (m, 2H), 1.80 (m, 2H), 1.29 (t, 3H). ¹³C NMR(CDCl₃-CD₃OD, 100 MHz) δ 151.9, 148.1, 135.8, 133.7, 130.2, 128.7,125.8, 125.4, 122.5, 121.2, 118.8, 112.1, 66.8, 64.0, 60.8, 46.8, 28.6,26.6, 14.5. HRMS calcd for [M+H]⁺ 315.1821. found 315.1839.

Example 3 (L1) Preparation of4-Amino-1-[2-(1,2-dipalmitoyl-sn-glycero-3-phospho)ethyl]-1H-imidazo[4,5-c]quinoline(Compound (I), R₁=H, Y=W=X=O, n=2, m=1, R₂=R₃=n-C₁₅H₃₁CO)

Compound L1 was prepared in 80% yield following the general proceduredescribed in Example 1 above: ¹H NMR (CDCl₃-CD₃OD, 400 MHz): δ 8.22 (s,1H), 8.16 (d, 1H), 7.41 (t, 1H); 7.21 (t, 1H), 6.92 (d, 1H), 5.26 (m,1H), 4.82 (bs, 2H), 4.67 (bs, 2H), 4.42 (dd, 1H), 4.20 (dd, 1H), 4.05(t, 2H), 3.14 (q, 1H), 2.31 (m, 4H), 1.59 (m, 4H), 1.25 (m, 48H), 0.88(m, 6H); ¹³C NMR (CDCl₃-CD₃OD, 100 MHz): δ 173.6, 173.2, 148.1, 145.8,134.5, 133.9, 129.3, 125.5, 124.5, 118.4, 112.3, 100.3, 77.2, 70.1,70.0, 63.5, 62.3, 45.9, 34.1, 33.9, 31.7, 29.5, 29.5, 29.3, 29.2, 29.1,29.1, 28.9, 28.9, 24.7, 24.7, 22.5, 13.9, 8.3. HRMS calcd for [M+H]⁺859.5714. found 859.5688.

Example 4 (L2) Preparation of4-Amino-1-[2-(1,2-dipalmitoyl-sn-glycero-3-phospho)ethyl]-2-butyl-1H-imidazo[4,5-c]quinoline(Compound (I), R₁=n-C₄H₉, Y=W=X=O, n=2, m=1, R₂=R₃=n-C₁₅H₃₁CO)

Compound L2 was prepared in 78% yield following the general proceduredescribed in Example 1 above: ¹H NMR (CDCl₃-CD₃OD, 400 MHz): δ 8.23 (bs,1H), 7.39 (t, 1H), 7.22 (bs, 1H), 6.93 (bs, 1H), 5.25 (m, 1H), 4.7 (bs,2H), 4.6 (bs, 2H), 4.42 (dd, 1H), 4.19 (dd, 1H), 4.04 (t, 2H), 3.06 (bs,2H) 2.32 (m, 4H), 1.96 (p, 2H) 1.59 (m, 6H) 1.26 (m, 48H), 1.07 (t, 3H),0.88 (m, 6H); ¹³C NMR (CDCl₃-CD₃OD, 100 MHz): δ 173.6, 173.2, 157.2,147.4, 135.2, 133.6, 128.8, 124.2, 123.6, 120.9, 118.2, 112.2, 77.2,70.0, 69.9, 63.2, 62.2, 46.3, 33.9, 33.7, 31.6, 29.3, 29.3, 29.3, 29.1,29.0, 28.95, 28.9, 28.8, 28.7, 28.6, 27.0, 24.5, 24.5, 22.3, 22.1, 13.6,13.4. HRMS: calcd for [M+H]⁺ 915.6340. found 915.6309.

Example 5 (L3) Preparation of4-Amino-1-[2-(1,2-dipalmitoyl-sn-glycero-3-phospho)ethyl]-2-ethoxymethyl-1H-imidazo[4,5-c]quinoline(Compound (I), R₁=CH₂OCH₂CH₃, Y=W=X=O, n=2, m=1, R₂=R₃=n-C₁₅H₃₁CO)

Compound L3 was prepared in 86% yield following the general proceduredescribed in Example 1 above: ¹H NMR (CDCl₃-CD₃OD, 400 MHz) δ 8.05 (bs,1H), 7.29 (t, 1H), 7.09 (bs, 1H), 6.78 (bs, 1H), 5.11 (m, 1H), 4.80 (bs,4H), 4.60 (bs, 2H), 4.28 (dd, 1H), 4.07 (dd, 1H), 3.90 (t, 2H), 3.54 (q,2H), 2.18 (m, 4H), 1.59 (m, 4H), 1.16 (m, 51H), 0.76 (m, 6H); ¹³C NMR(CDCl₃-CD₃OD, 100 MHz): δ 173.4, 173.0, 153.3, 148.2, 135.7, 134.7,129.1, 124.4, 124.2, 121.1, 119.1, 112.8, 77.2, 70.2, 70.2, 66.6, 65.4,64.2, 63.5, 62.5, 57.7, 47.1, 45.7, 34.3, 34.1, 31.9, 29.7, 29.7, 29.6,29.5, 29.3, 29.3, 29.1, 29.1, 24.9, 22.7, 15.0, 14.1, 8.6. HRMS calcdfor [M+H]⁺ 917.6132. found 917.6162.

Example 6 (L4) Preparation of4-Amino-1-[2-(1,2-dipalmitoyl-sn-glycero-3-phospho)butyl]-2-ethoxymethyl-1H-imidazo[4,5-c]quinoline(Compound (I), R₁=H, Y=W=X=O, n=4, m=1, R₂=R₃=n-C₁₅H₃₁CO)

Compound L4 was prepared in 26% yield following the general proceduredescribed in Example 1 above: ¹H NMR (CDCl₃, 400 MHz): δ 11.2 (bs, 1H),7.78 (d, 1H), 7.30 (t, 1H), 7.20 (d, 1H), 6.78 (t, 1H), 6.39 (bs, 1H),5.28 (m, 1H), 4.79 (s, 2H), 4.43-4.50 (m, 3H), 4.11-4.27 (m, 5H), 3.67(dd, 2H), 2.41 (bs, 2H), 2.30 (dd, 4H), 1.96 (bs, 1H), 1.60 (m, 4H),1.25 (m, 54H), 0.88 (1, 6H); ¹³C NMR (CDCl₃, 100 MHz): δ 173.4, 173.0,150.9, 148.9, 134.7, 134.2, 128.0, 124.3 (2), 120.5, 118.4, 111.8, 70.3,70.2, 66.8, 64.7, 64.4, 64.3, 63.4 (2), 62.4, 46.6, 34.2, 34.1, 31.9,29.6 (3), 29.4, 29.3 (2), 29.2, 29.1, 28.2, 27.4, 24.8 (2), 22.6, 15.1,14.1. HRMS calcd for [M−H]⁻ 943.6289. found 943.6251.

Example 7 General procedure for the preparation of4-Amino-1-[2-(1,2-dipalmitoyl-sn-glycero-3-diphospho)alkyl]-1H-imidazo[4,5-c]quinolines(Compound (I), Y=W=X=O, m=2)

Imidazoquinoline diphosphate diglycerides VIII were prepared by couplingthe imidazoquinoline monophosphomorpholidate VI, prepared in crude formfrom imidazoquinoline III, with 1,2-diacyl-sn-glycerol-3-phosphatesodium salt VII according to methods known in the art (Biochim. Biophys.Acta 1980, 619, 604, J. Biol. Chem., 1990, 265(11), (6112-6117) J. Org.Chem. 1997, 62, 2144-2147) as follows: POCl₃ (2.0 eq) andimidazoquinoline III (1.0 eq) were added to trimethyl phosphate (0.38 M)at 0° C. After stirring 15 h at 0° C., the reaction mixture waspartitioned between H₂O and Et₂O and the layers separated. The organiclayer was extracted three times with H₂O and the pH of combined aqueouslayers was adjusted to pH 9 with aq NH₄OH. The aqueous solution wasconcentrated and dried under high vacuum and the residue obtainedpurified by chromatography on silica gel with CHCl₃-MeOH—H₂O-Et₃N(gradient elution, 90:10:0.5:0.5→60:40:5:1). The product obtained wasdissolved in dioxane (O.12M) at 50° C. and treated with 4N HCl (1.5 eq).The HCl salt that precipitated was collected, rinsed with dioxane, anddried under high vacuum. Morpholine (5.0 eq) was added to a suspensionof the salt in 1:1 t-BuOH—H₂O (0.5M) and the reaction mixture was heatedto 90° C. and treated with a solution of 1,3-dicyclohexylcarbodiimide(DCC, 5.0 eq) in t-BuOH (0.33 M). After 1 h at 90° C., the cooledreaction mixture was partitioned between H₂O and Et₂O and the layersseparated. The organic layer was extracted twice with H₂O and thecombined aqueous layers concentrated and dried under high vacuum. Asuspension of the crude phosphomorpholidate VI obtained (1.5 eq) and VII(1.0 eq) in a small volume of pyridine was concentrated under vacuum,and then co-evaporated twice with toluene, and dried under high vacuum;this procedure was repeated twice more. 4,5-Dicyanoimidazole (DCI, 3.0eq) was then added to a suspension of the dried solids in pyridine (0.10M) and the reaction mixture was stirred at room temperature for 10 days.The resulting mixture was concentrated and the residue obtainedpartitioned between H₂O—CH₂Cl₂ and the layers separated. The aqueouslayer was extracted twice with CH₂Cl₂ and the combined organic layerswere dried (Na₂SO₄), and concentrated. Chromatography on silica gel withCHCl₃-MeOH—H₂O (gradient elution, 90:10:0.5→70:30:2) afforded compoundVIII as a colorless solid.

Example 8 (L5) Preparation of4-Amino-1-[2-(1,2-dipalmitoyl-sn-glycero-3-diphospho)ethyl]-2-ethoxymethyl-1H-imidazo[4,5-c]quinoline(Compound (I), R₁=CH₂OCH₂CH₃, Y=W=X=O, n=2, m=2, R₂=R₃=n-C₁₅H₃₁CO)

Compound L5 was prepared in 22% yield following the general proceduredescribed in Example 6 above: ¹H NMR (CDCl₃, 400 MHz): δ 8.17 (bs, 1H),7.10-7.40 (2-3 m, 2-3H), 5.25 (bs, 1H), 4.60-5.00 (bm, 3H), 4.38 (m,1H), 4.05-4.22 (m, 3H), 3.60-3.82 (m, 4H), 3.41 (bs, 1H), 3.10 (dd, 2Hof Et₃N), 2.28 (m, 4H), 1.84 (dd, 1H), 1.56 (m, 5H), 1.25 (m, 54H), 0.88(t, 7H); ¹³C NMR (CDCl₃, 100 MHz): δ 173.5, 173.1, 152.4, 147.7, 135.8,134.2, 128.8, 124.5, 123.6, 122.0, 118.8, 112.2, 77.2, 70.0, 68.1, 66.5,63.8, 62.4, 54.6, 46.5, 45.5, 38.5, 33.9, 33.0, 29.5, 29.4, 29.1, 28.9,28.7, 25.0, 24.6, 23.5, 22.7, 22.5, 14.6, 13.8, 13.7, 13.2, 10.7, 8.1.HRMS calcd for [M+H]⁺ 997.5796. found 997.5776.

Example 9 In Vivo Testing of Lipidated TLR7/8

TLR7/8 ligands may promote various aspects of the immune response inmice, noticeably the CD8 response. The difference in response between aTLR7/8 ligand (the “core” compound), and its corresponding lipidatedderivative are investigated using techniques such as those describedbelow.

Formulation of the compounds for a comparison study requires taking intoaccount the different molecular weights of the core and lipidatedmolecules, (e.g. 45 and 4.5 μg of the lipidated compound L3 correspondsto approximately to 15 and 1.5 μg of the corresponding core compound,“L3 core”), allowing for the side by side comparison of thecorresponding groups in the study. Higher doses of L3 (200 μg) and of L3core (150 μg) are also tested. In one such study formulations summarizedand described below (table 1) are used to vaccinate 6-8 week old C57BL/6(H2Kb), female mice (10/group). The mice receive two injections 14 daysapart and are bled during weeks 1, 3 and 4 (for precise bleed days seeFIG. 1). The mice are vaccinated intramuscularly. A heterologousprime/boost using recombinant adenovirus coding for the SIV-p27 proteinand adjuvanted p27 are used as control groups, the adenovirus isinjected at a dose of 5×10⁸ VP. The study design is represented in FIG.1.

TABLE 1 Summary of the formulations description p27 QS21 MPL SB62c^(b)L3 L3 Core QS/MPL (liposome based 5 5 5 — — — formulation) QS/MPL + 200μg L3 5 5 5 — 200 — QS/MPL + 45 μg L3 5 5 5 — 45 — QS/MPL + 4.5 μg L3 55 5 — 4.5 — QS/MPL + 150 μg L3 core 5 5 5 — — 150 QS/MPL + 15 μg L3 core5 5 5 — — 15 QS/MPL + 1.5 μg L3 core 5 5 5 — — 1.5 QS/MPL/SB62C(emulsion- 5 5 — 5 μl based formulation) QS/MPL/SB62C + 200 μg L3 5 5 55 μl 200 — QS/MPL/SB62C + 45 μg L3 5 5 5 5 μl 45 — QS/MPL/SB62C + 4.5 μgL3 5 5 5 5 μl 4.5 — QS/MPL/SB62C + 150 μg 5 5 5 5 μl — 150 L3 coreQS/MPL/SB62C + 15 μg L3 5 5 5 5 μl — 15 core QS/MPL/SB62C + 1.5 μg L3 55 5 5 μl — 1.5 core p27 5 — — — — — naive — — — — — — All compounds arein μg unless otherwise stated. ^(b)SB62c contains the oil in water SB62and cholesterol

In the study design molecules are formulated in either a liposome-basedor an oil-in-water-based adjuvant composition containing QS21 and MPLimmunostimulants. To assess the added value of TLR7/8L, the innate andadaptive immune responses induced by the formulations containing theTLR7/8L, QS21 and MPL are compared to the one induced by thecorresponding QS21 and MPL containing formulations.

The induction of antigen-specific CD8 and CD4 responses is assessed bymeasuring intracellular cytokines 7 days after the second injection.Peripheral blood lymphocytes (PBLs) are stimulated in the presence of apool of peptides encompassing the whole p27 antigen (15-mers peptides,overlap by 11). The secretion of cytokines is blocked by Brefeldin A andthe presence of 3 cytokines (IFNγ, TNFα and IL2) is evaluated by flowcytometry after intracellular staining with appropriate antibodies.

Studies similar to that described above were performed using CRX-642 andits lipidated counterpart L3. FIGS. 2 and 3 show the p27-specificT-cells frequency observed 7 days after the second immunization. In adose response manner, the p27-specific CD8 frequency was clearlyincreased when liposomes containing L3 were co-administered with MPL andQS-21 formulations as compared to the control formulation withoutTLR7/8L (FIG. 2). Noticeably both the liposome-based formulation and theoil-in-water based formulations allowed for an increased in the CD8response in presence of the lipidated TLR7/8L as compared to thecorresponding control formulations without TLR7/8L. Furthermore theability of generating cytokine-producing CD8 T cells was dependent onthe lipidated nature of the TLR7/8 ligand, as the core molecule L3 corein contrast to L3 did not increase the response.

In complement to the evaluation of the induced CD8 response,antigen-specific cytotoxic activity may be assessed in vivo. Briefly,targets pulsed with p27 peptides spanning the whole protein and controlunpulsed targets are injected into immunized mice and 24 hrs afterinjection, the p27-specific cytotoxicity is assessed by thedisappearance of the pulsed target.

Complementary cytotoxic activity studies were performed with theevaluation of induced CD8 response of L3 core and L3 explained above. Ahigher cytotoxic activity was detected in mice immunized with lipidatedTLR7/8 ligand-based formulations than with in mice receiving a coreTLR7/8 ligand-based formulations (FIG. 3). This activity was higher thanthe one induced by control formulations based on QS21 and MPL only,especially when high doses of lipidated TLR7/8L were used.

As shown for the CD8 response, the p27-specific CD4 frequency increasedwhen liposomes containing L3 were co-administered with theliposome-based MPL and QS-21-containing formulation as compared to thecontrol formulation, the response being dependent on the dose TLR7/8Linjected (FIG. 4). As for the CD8 response, the core molecule L3 corewas not able to induce a CD4 response over the one induced by thecontrol formulations.

When administered in an emulsion-based formulation, the lipidatedTLR7/8L was also able to increase the CD4 T-cell response over the levelreached by the control formulation.

When lipidated, the added value of the TLR7/8 ligands within differentformulations is shown by the increase up to 5-fold in cytokine-producingT cell frequency (both CD8 and CD4 T cells). Interestingly the cytokineprofile of T-cell response was characterized by the high frequency ofdouble positive T-cells (IFNγ⁺ TNα⁺).

Additional investigation illustrates the ability of lipidated TLR7/8compounds to induce innate chemokines and pro-inflammatory cytokinesamong which type I IFN is known to be required for the programming ofnaïve CD8-T-cell (survival, differentiate and memory development). Thesecytokines are measured in the sera of mice 3 and 24 hrs after the firstinjection (FIGS. 5 and 6).

Results of cytokine profiles for L3 core and L3 show similar profile ofcytokines are observed between the liposome-based and emulsion-basedformulations. TLR7/8 ligands are known to induce IFNα due to theirability to stimulate plasmacytoid dendritic cells and IFNα was indeeddetected in the serum of mice immunized with L3 in a dose-dependentmanner and at higher level than for its core counterpart, L3 core. LowIL-12p70 production, close to background level, was also detected. Levelof INFγ increased at low dose of L3 while other inflammatory cytokinessuch as TNFα or IL-6 were enhanced when both L3 core and L3 were addedto QS21 and MPL. The chemokines MCP-1 and MIG were both increased up to10 fold with both compounds. Altogether, these data show that the testedlipidated molecule is as effective if not more potent than thecorresponding core molecules to induce cytokine production in vivo.

The invention claimed is:
 1. A compound comprising Formula II

wherein R₁=H, n-butyl, ethoxymethyl n=2-4 X=O Y=O W=O m=1-2, R₂=hexadecanoyl, and R₃=hexadecanoyl.
 2. The compound of claim 1 wherein the compound is:


3. The compound of claim 1 wherein the compound is:


4. The compound of claim 1 wherein the compound is:


5. The compound of claim 1 wherein the compound is:


6. The compound of claim 1 wherein the compound is: 